Crystalline form of a benzimidazole-carboxamide medicinal compound

ABSTRACT

The invention provides crystalline forms of the novel benzimidazole-carboxamide 5-HT 4  receptor agonist compound, 4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)amino]methyl}-piperidin-1-ylmethyl)piperidine-1-carboxylic acid methyl ester. The invention also provides pharmaceutical compositions comprising the crystalline compound, methods of using the compound to treat diseases associated with 5-HT 4  receptor activity, and processes useful for preparing crystalline forms of the compound.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/684,478, filed on May 25, 2005, the disclosure of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is directed to crystalline forms of abenzimidazole-carboxamide compound which is useful as a 5-HT₄ receptoragonist. The invention is also directed to pharmaceutical compositionscomprising the crystalline compound, methods of using the compound fortreating or preventing medical conditions mediated by 5-HT₄ receptoractivity, and processes useful for preparing the compound in crystallineform.

2. State of the Art

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter that iswidely distributed throughout the body, both in the central nervoussystem and in peripheral systems. At least seven subtypes of serotoninreceptors have been identified and the interaction of serotonin withthese different receptors is linked to a wide variety of physiologicalfunctions. There has been, therefore, substantial interest in developingtherapeutic agents that target specific 5-HT receptor subtypes.

In particular, characterization of 5-HT₄ receptors and identification ofpharmaceutical agents that interact with them has been the focus ofsignificant recent activity. (See, for example, the review by Langloisand Fischmeister, J. Med. Chem. 2003, 46, 319-344.) 5-HT₄ receptoragonists are useful for the treatment of disorders of reduced motilityof the gastrointestinal tract. Such disorders include irritable bowelsyndrome (IBS), chronic constipation, functional dyspepsia, delayedgastric emptying, gastroesophageal reflux disease (GERD), gastroparesis,post-operative ileus, intestinal pseudo-obstruction, and drug-induceddelayed transit. In addition, it has been suggested that some 5-HT₄receptor agonist compounds may be used in the treatment of centralnervous system disorders including cognitive disorders, behavioraldisorders, mood disorders, and disorders of control of autonomicfunction.

Despite the broad utility of pharmaceutical agents modulating 5-HT₄receptor activity, few 5-HT₄ receptor agonist compounds are in clinicaluse at present. Accordingly, there is a need for new 5-HT₄ receptoragonists that achieve their desired effects with minimal side effects.Preferred agents may possess, among other properties, improvedselectivity, potency, pharmacokinetic properties, and/or duration ofaction.

To effectively use such agents, it is desirable that the 5-HT₄ receptoragonist compounds be provided in a crystalline form that can be readilymanufactured and that has acceptable physical and chemical stability.

SUMMARY OF THE INVENTION

The invention provides crystalline forms of a novel compound thatpossesses 5-HT₄ receptor agonist activity. Among other properties, thecompound of the invention has been found to be a potent and selective5-HT₄ receptor agonist. In addition, the compound of the invention hasbeen found to exhibit favorable pharmacokinetic properties which arepredictive of good bioavailability upon oral administration.

Accordingly, the invention provides crystalline forms of the compoundhaving the chemical structure:

or a solvate thereof. The above compound (hereinafter compound 1) isdesignated4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)amino]methyl}-piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester, according to the AutoNom software, provided by MDLInformation Systems, GmbH (Frankfurt, Germany). The fused ring structure“benzoimidazole” is alternatively named “benzimidazole”. The two termsare equivalent as used herein.

A crystalline form of4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)amino]methyl}-piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester has been found to have a melting temperature in therange of about 145° C. to about 155° C., typically between about 146 andabout 148° C., a degradation temperature above about 240° C., and toexhibit weight changes of less than about 0.25% when exposed to a rangeof relative humidity between about 2% and about 90% at room temperature.Additional crystalline forms of4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)amino]methyl}-piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester are also provided in further aspects of the invention.

Among other uses, the crystalline forms of compound 1 are expected to beuseful for preparing pharmaceutical compositions for treating disordersof reduced motility of the gastrointestinal tract. Accordingly, inanother of its composition aspects, the invention provides apharmaceutical composition comprising a pharmaceutically-acceptablecarrier and crystalline4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)amino]methyl}-piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester or a solvate thereof.

In addition, the invention provides a method of treating a disease orcondition associated with 5-HT₄ receptor activity, e.g. a disorder ofreduced motility of the gastrointestinal tract, the method comprisingadministering to the mammal, a therapeutically effective amount of thecrystalline compound of the invention.

In another method aspect, the invention provides processes for preparingcompound 1 in crystalline form.

The invention also provides the crystalline compound of the invention asdescribed herein for use in medical therapy, as well as the use of thecrystalline compound of the invention in the manufacture of aformulation or medicament, especially for the manufacture of amedicament for treating a disorder of reduced motility of thegastrointestinal tract in a mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of the present invention are illustrated by reference tothe accompanying drawings.

FIG. 1 shows a powder x-ray diffraction (PXRD) pattern of crystalline4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)amino]methyl}-piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester (Form I).

FIG. 2 shows a differential scanning calorimetry (DSC) trace (top trace,right vertical axis) and a thermal gravimetric analysis (TGA) trace(bottom trace, left vertical axis) for crystalline4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)amino]methyl}-piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester (Form I).

FIG. 3 shows a dynamic moisture sorption (DMS) isotherm for crystalline4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)amino]methyl}-piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester (Form I).

FIG. 4 shows a differential scanning calorimetry (DSC) trace (top trace,right vertical axis) and a thermal gravimetric analysis (TGA) trace(bottom trace, left vertical axis) for crystalline4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)amino]methyl}-piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester (Form II).

FIG. 5 shows a powder x-ray diffraction (PXRD) pattern of crystalline4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)amino]methyl}-piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester (Form III).

FIG. 6 shows a differential scanning calorimetry (DSC) trace (top trace,right vertical axis) and a thermal gravimetric analysis (TGA) trace(bottom trace, left vertical axis) for crystalline4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)amino]methyl}-piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester (Form III).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides crystalline forms of a novelbenzimidazole-carboxamide 5-HT₄ receptor agonist.

Definitions

When describing the compound, compositions and methods of the invention,the following terms have the following meanings, unless otherwiseindicated.

The term “compound” means a compound that was synthetically prepared orproduced in any other way, such as by metabolism.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.

The term “treatment” as used herein means the treatment of a disease,disorder, or medical condition in a patient, such as a mammal(particularly a human) which includes:

-   -   (a) preventing the disease, disorder, or medical condition from        occurring, i.e., prophylactic treatment of a patient;    -   (b) ameliorating the disease, disorder, or medical condition,        i.e., eliminating or causing regression of the disease,        disorder, or medical condition in a patient;    -   (c) suppressing the disease, disorder, or medical condition,        i.e., slowing or arresting the development of the disease,        disorder, or medical condition in a patient; or    -   (d) alleviating the symptoms of the disease, disorder, or        medical condition in a patient.

The term “pharmaceutically-acceptable salt” means a salt prepared froman acid or base which is acceptable for administration to a patient,such as a mammal. Such salts can be derived frompharmaceutically-acceptable inorganic or organic acids and frompharmaceutically-acceptable bases. Typically,pharmaceutically-acceptable salts of compounds of the present inventionare prepared from acids.

Salts derived from pharmaceutically-acceptable acids include, but arenot limited to, acetic, adipic, benzenesulfonic, benzoic,camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic,hydrobromic, hydrochloric, lactic, maleic, malic, mandelic,methanesulfonic, mucic, nitric, pantothenic, phosphoric, succinic,sulfuric, tartaric, p-toluenesulfonic, xinafoic (1-hydroxy-2-naphthoicacid), naphthalene-1,5-disulfonic acid and the like.

The term “solvate” means a complex or aggregate formed by one or moremolecules of a solute, i.e. a compound of the invention or apharmaceutically-acceptable salt thereof, and one or more molecules of asolvent. Such solvates are typically crystalline solids having asubstantially fixed molar ratio of solute and solvent. Representativesolvents include by way of example, water, methanol, ethanol,isopropanol, acetic acid, and the like. When the solvent is water, thesolvate formed is a hydrate.

The term “amino-protecting group” means a protecting group suitable forpreventing undesired reactions at an amino nitrogen. Representativeamino-protecting groups include, but are not limited to, formyl; acylgroups, for example alkanoyl groups, such as acetyl; alkoxycarbonylgroups, such as tert-butoxycarbonyl (Boc); arylmethoxycarbonyl groups,such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc);arylmethyl groups, such as benzyl (Bn), trityl (Tr), and1,1-di-(4′-methoxyphenyl)methyl; silyl groups, such as trimethylsilyl(TMS) and tert-butyldimethylsilyl (TBDMS); and the like.

Synthetic Procedures

Compound 1,4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)amino]methyl}-piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester, can be prepared from readily available startingmaterials using the following general procedure. Although a particularaspect of the present invention is illustrated in the schemes below,those skilled in the art will recognize that all aspects of the presentinvention can be prepared using the methods described herein or by usingother methods, reagents and starting materials known to those skilled inthe art. It will also be appreciated that where typical or preferredprocess conditions (i.e., reaction temperatures, times, mole ratios ofreactants, solvents, pressures, etc.) are given, other processconditions can also be used unless otherwise stated. Optimum reactionconditions may vary with the particular reactants or solvent used, butsuch conditions can be determined by one skilled in the art by routineoptimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. The choice of asuitable protecting group for a particular functional group, as well assuitable conditions for protection and deprotection, are well known inthe art. For example, numerous protecting groups, and their introductionand removal, are described in T. W. Greene and G. M. Wuts, ProtectingGroups in Organic Synthesis, Third Edition, Wiley, New York, 1999, andreferences cited therein.

The compound of the invention may be prepared by reacting apiperidinylmethyl-carboxylic acid amide intermediate (II):

with formylpiperidinyl intermediate (III):

The reaction is typically conducted by contacting (II) with betweenabout 1 and about 2 equivalents of intermediate (III) in an inertdiluent in the presence of between about 1 and about 2 equivalents of areducing agent. Optionally, about one equivalent of a weak acid, such asacetic acid can be included to accelerate the reaction. The reaction maybe conducted at a temperature between about 0° C. and about 30° C.,typically between about 20° C. and about 30° C., for about 0.25 to about2 hours, or until the reaction is substantially complete. The product isisolated by standard procedures.

Suitable inert diluents include dichloromethane, N,N-dimethylformamidetrichloromethane, 1,1,2,2-tetrachloroethane, and the like. Typicalreducing agents include sodium triacetoxyborohydride, sodiumborohydride, and sodium cyanoborohydride. When the amine (II) issupplied as an acid salt, typically between about 1 and about 3equivalents of an amine base, such as N,N-diisopropylethylamine,triethylamine, pyridine, and the like, is included in the reaction.

The piperidinylmethyl-carboxylic acid amide intermediate (II) isprepared from readily available starting materials by the procedureillustrated in Scheme A.

where P¹ represents an amino protecting group, such astert-butoxycarbonyl (Boc).

First, the carboxylic acid (IV) is reacted with a protected aminomethylpiperidine to form a protected intermediate (V). This reaction istypically conducted by contacting (IV) with between about 1 and about 2equivalents of protected aminomethylpiperidine in a polar aproticdiluent, in the presence of an amide coupling agent, for exampleN-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)combined with hydroxybenzotriazole (HOBt) or 1,1′-carbonyldiimidazole(CDI) combined with 1,4-diazabicyclo[2,2,2]octane (DABCO). The reactionis typically conducted at a temperature in the range of about 0° C. toabout 60° C. for between about 1 and about 24 hours or until thereaction is substantially complete.

The protecting group P¹ is removed from intermediate (V) by conventionalmeans to provide intermediate (II). For example, when Boc is used as theprotecting group, it may be removed by treatment with an acid, such astrifluoroaacetic acid or hydrochloric acid.

The carboxylic acid (IV) can be prepared from a diaminobenzoic acid orester by the process illustrated in Scheme B:

where R represents methyl or hydrogen. Intermediate (VII) is reactedwith isobutyric acid to form the acid intermediate (IV). This reactionis typically conducted by contacting the acid or ester (VII) withbetween about 2 and about 4 equivalents of isobutyric acid in an aqueousacidic solution. The reaction is typically conducted at a temperature inthe range of about 80° C. to about 100° C. for about 12 to about 72hours. The pH of the solution is then raised by the addition of base,such as sodium hydroxide, and the product isolated by conventionalmeans.

A convenient process for providing intermediate (VII) as the methylester uses 2-amino-3-nitrobenzoic acid methyl ester (VI):

as the starting material. Typically, 2-amino-3-nitrobenzoic acid methylester (VI) is dissolved in a polar diluent and reduced by exposure to ahydrogen atmosphere in the presence of a transition metal catalyst toprovide the diaminobenzoic acid methyl ester (VII). The reaction istypically conducted at ambient temperature for about 12 to about 72hours.

A process for preparing the formylpiperidinyl intermediate (III) isillustrated in Scheme C.

First 4-hydroxymethylpiperidine is reacted with methylchloroformate toform the hydroxymethylpiperidine intermediate (VIII). The reaction istypically conducted by contacting 4-hydroxymethylpiperidine in anaqueous solution with between about 3 and about 5 equivalents ofmethylchloroformate in the presence of between about 3 and about 5equivalents of base. The reaction is typically conducted at atemperature in the range of about 0° C. to about 30° C. for about 12 toabout 72 hours or until the reaction is substantially complete.Intermediate (VIII) is then oxidized to form the formylpiperidinylintermediate (III). The oxidation reaction typically makes use of anoxidation reagent such as a combination of oxalyl chloride anddimethylsulfoxide (Swern oxidation), a chromate reagent, such aspyridinium chlorochromate, or an oxidizing agent, such as sodiumhypochlorite, together with a catalyst such as2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO).

Further details regarding specific reaction conditions and otherprocedures for preparing compound 1 or intermediates thereto aredescribed in the examples below and in the commonly-assigned U.S.Provisional Application No. 60/684,478, and in U.S. application Ser. No.______, filed on even date herewith (Attorney Docket No. P-212-US1),which is incorporated herein by reference.

Crystalline Forms

The invention provides4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)amino]methyl}-piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester (compound 1) in crystalline freebase form or a solvatethereof. Three distinguishable forms of crystalline4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)amino]methyl}-piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester have been observed.

Crystalline Form I of the present invention is a crystalline freebase.Form I is characterized by a powder x-ray diffraction (PXRD) patternhaving two or more diffraction peaks at 2θ values selected from15.08±0.20, 15.41±0.20, 19.00±0.20, 19.70±0.20, and 23.68±0.20. Inparticular, Form I is characterized by a powder x-ray diffractionpattern having diffraction peaks at 2θ values of 19.00±0.20 and19.70±0.20.

As is well known in the field of powder x-ray diffraction, peakpositions of PXRD spectra are relatively less sensitive to experimentaldetails, such as details of sample preparation and instrument geometry,than are the relative peak heights. Thus, in one aspect, the crystallineForm I is characterized by a powder x-ray diffraction pattern in whichthe peak positions are substantially in accordance with those shown inFIG. 1.

Form I has been further characterized by x-ray diffraction analysis ofcrystal structure, providing the following lattice parameters: unit cellis orthorhombic with dimensions a=16.9053 Å, b=9.5172 Å, c=15.4659 Å;space group is Pna2₁; calculated density is 1.22 g/cm³. The resultingmolecular structure confirms the chemical composition is that ofcompound 1 and that the assymetric unit does not contain water or othersolvent molecules. Powder x-ray diffraction peaks predicted from thederived atomic positions are in excellent agreement with observedresults.

Crystalline Form I of the present invention is also characterized byhigh temperature thermal stability as evidenced by its differentialscanning calorimetry (DSC) profile which exhibits a peak in endothermicheat flow in the range of about 145° C. to about 155° C., typicallybetween about 146 and 148° C., as illustrated in FIG. 2, which may beidentified as a melting point. Furthermore, the thermal gravimetricanalysis (TGA) profile shows no significant weight change event belowthe onset of degradation which is above about 240° C.

In yet another aspect, the present crystalline form is characterized byits infrared absorption spectrum which shows significant absorptionbands at about 766, 1097, 1251, 1413, 1449, 1579, 1609, 1640, and 1696cm⁻¹.

The present crystalline form has been demonstrated to have a reversiblesorption/desorption profile with an exceptionally low level ofhygroscopicity (i.e., less than about 0.25% weight gain in the humidityrange of 2% relative humidity to 90% relative humidity at roomtemperature) as shown in FIG. 3.

Additionally, crystalline Form I of compound 1 has been found to bestable upon exposure to elevated temperature and humidity. After storagefor three months at 40° C. and 75% relative humidity, there were nodetectable changes in the DSC, TGA, or PXRD profiles, in the chemicalpurity as determined by HPLC analysis, and no observable changes invisual appearance. There were also no changes detectable by DSC, TGA, orPXRD after milling particles of Form I from a volume-based mean particlesize of about 470 microns to a volume-based mean particle size of about15, about 22, or about 29 microns.

Crystalline Form II of compound 1 is characterized by the DSC and TGAprofiles of FIG. 4. TGA analysis shows an onset of weight loss in thetemperature range of about 95 to about 105° C., typically at about 100°C., in a step profile consistent with water or solvent loss, while theDSC profile exhibits a peak in endothermic heat flow at between about143 and about 145° C., concurrent with the melting event. The PXRDpattern of Form II is indistinguishable from that of Form I. Whilepositive identification has not been made, the TGA profile of Form II isconsistent with the interpretation of Form II as a solvate.

The third crystalline form of the invention has been identified as amonohydrate. Form III is characterized by a powder x-ray diffraction(PXRD) pattern having two or more diffraction peaks at 2θ valuesselected from 9.14±0.20, 12.41±0.20, 12.74±0.20, 17.75±0.20, 18.47±0.20,20.63±0.20, 21.13±0.20, and 27.05±0.20, as illustrated in FIG. 5. Inparticular, Form III is characterized by a powder x-ray diffractionpattern having diffraction peaks at 2θ values of 9.14±0.20, and20.63±0.20.

The DSC and TGA profiles of Form III, shown in FIG. 6, demonstrate thematerial undergoes a step profile weight loss with onset in thetemperature range of about 65 to about 75° C., typically at about 70°C., and a peak in endothermic heat flow at a temperature between about90 and about 100° C., consistent with loss of the monohydrate water andsubsequent melting. Form III has been further characterized by x-raydiffraction analysis of crystal structure, providing the followinglattice parameters: unit cell is monoclinic with dimensions a=14.8101 Å,b=9.9985 Å, c=17.9222 Å; β=106.3020°, space group is P2₁/n; calculateddensity is 1.23 g/cm³. The resulting molecular structure confirms thechemical composition is that of compound 1 and that the assymetric unitcontains a single water molecule.

The separate crystalline forms of the present invention may bereproducibly obtained by the following procedures. Crystalline Form Imay be prepared by dispersing compound 1 in an inert diluent selectedfrom acetonitrile, ether, cyclohexane, and ethyl acetate in a proportionof between about 15 mg and about 25 mg compound 1 per milliliter ofdiluent to form a mixture, and allowing the mixture to evaporate atambient temperature, resulting in crystal formation.

Alternatively, Form I may be obtained by a solvent exchange process fromcrude compound 1 in solution, without first isolating amorphous compound1, as described in Example 3 below. Typically the reaction to preparecompound 1 is carried out in a polar aprotic diluent, such asdichloromethane, in which the compound is highly soluble. To preparecrystalline Form I, acetonitrile is added while vacuum distilling thecrude reaction product to remove the dichloromethane. A mixture havingbetween about 1 and about 200 mg compound 1 per milliliter ofacetonitrile, typically between about 50 and about 125 mg compound 1 permilliliter of acetonitrile, is prepared from the residue remaining afterdistillation and heated to a temperature sufficient to dissolve theresidue, for example a temperature of about 75° C. The mixture is thencooled to a temperature of no more than about 20° C. to providecrystalline Form I, which is isolated by conventional procedures. In anexemplary process, the mixture is cooled until nucleation occurs,typically at a temperature of between about 55 and about 65° C. and heldat that temperature for about an hour. It is then cooled at a rate ofbetween about 0.25 and about 0.4° C. per minute to a temperature ofabout 20° C. To increase the yield of crystalline Form I, the mixturemay be further cooled at a rate of between about 0.5 and about 0.75° C.per minute to a temperature of between about 0 and about 5° C.

To prepare Form II, amorphous compound 1 is dispersed in hexane atambient temperature to a final concentration of about 10 mg/mL and theresulting mixture is sonicated. After about 24 hours at ambienttemperature, crystalline solids of Form II are obtained.

Form III is prepared by dissolving amorphous compound 1 in a 1:1ethanol:water solvent mixture at ambient temperature to a finalconcentration of about 20 mg/mL and sonicating the solution for about 30seconds. The solution is allowed to partially evaporate in an uncappedvial. After about 24 hours, crystalline solids of Form III are obtained.

Pharmaceutical Compositions

The crystalline benzimidazole-carboxamide compound of the invention istypically administered to a patient in the form of a pharmaceuticalcomposition. Such pharmaceutical compositions may be administered to thepatient by any acceptable route of administration including, but notlimited to, oral, rectal, vaginal, nasal, inhaled, topical (includingtransdermal) and parenteral modes of administration.

Accordingly, in one of its compositions aspects, the invention isdirected to a pharmaceutical composition comprising apharmaceutically-acceptable carrier or excipient and a therapeuticallyeffective amount of a crystalline form of compound 1. Optionally, suchpharmaceutical compositions may contain other therapeutic and/orformulating agents if desired.

The pharmaceutical compositions of the invention typically contain atherapeutically effective amount of the compound of the presentinvention. Typically, such pharmaceutical compositions will contain fromabout 0.1 to about 95% by weight of the active agent. i.e. the presentcompound; including, from about 5 to about 70% by weight; and from about10 to about 60% by weight of the active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable pharmaceutical composition for a particular mode ofadministration is well within the scope of those skilled in thepharmaceutical arts. Additionally, the ingredients for such compositionsare commercially-available from, for example, Sigma, P.O. Box 14508, St.Louis, Mo. 63178. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following: (1)sugars, such as lactose, glucose and sucrose; (2) starches, such as cornstarch and potato starch; (3) cellulose, such as microcrystallinecellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5)malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter andsuppository waxes; (9) oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10)glycols, such as propylene glycol; (11) polyols, such as glycerin,sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyloleate and ethyl laurate; (13) agar; (14) buffering agents, such asmagnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19)ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxiccompatible substances employed in pharmaceutical compositions.

The pharmaceutical compositions of the invention are typically preparedby thoroughly and intimately mixing or blending a compound of theinvention with a pharmaceutically-acceptable carrier and one or moreoptional ingredients. If necessary or desired, the resulting uniformlyblended mixture can then be shaped or loaded into tablets, capsules,pills and the like using conventional procedures and equipment.

The pharmaceutical compositions of the invention are preferably packagedin a unit dosage form. The term “unit dosage form” refers to aphysically discrete unit suitable for dosing a patient, i.e., each unitcontaining a predetermined quantity of active agent calculated toproduce the desired therapeutic effect either alone or in combinationwith one or more additional units. For example, such unit dosage formsmay be capsules, tablets, pills, and the like.

In a preferred embodiment, the pharmaceutical compositions of theinvention are suitable for oral administration. Suitable pharmaceuticalcompositions for oral administration may be in the form of capsules,tablets, pills, lozenges, cachets, dragees, powders, granules; or as asolution or a suspension in an aqueous or non-aqueous liquid; or as anoil-in-water or water-in-oil liquid emulsion; or as an elixir or syrup;and the like; each containing a predetermined amount of a compound ofthe present invention as an active ingredient.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills and the like), the pharmaceutical compositionsof the invention will typically comprise a compound of the presentinvention as the active ingredient and one or morepharmaceutically-acceptable carriers, such as sodium citrate ordicalcium phosphate. Optionally or alternatively, such solid dosageforms may also comprise: (1) fillers or extenders, such as starches,microcrystalline cellulose, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as carboxymethylcellulose, alginates,gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants,such as glycerol; (4) disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and/or sodium carbonate; (5) solution retarding agents, such asparaffin; (6) absorption accelerators, such as quaternary ammoniumcompounds; (7) wetting agents, such as cetyl alcohol and/or glycerolmonostearate; (8) absorbents, such as kaolin and/or bentonite clay; (9)lubricants, such as talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and/or mixtures thereof;(10) coloring agents; and (11) buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants can also be presentin the pharmaceutical compositions of the invention. Examples ofpharmaceutically-acceptable antioxidants include: (1) water-solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfate sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal-chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like. Coating agents fortablets, capsules, pills and like, include those used for entericcoatings, such as cellulose acetate phthalate (CAP), polyvinyl acetatephthalate (PVAP), hydroxypropyl methylcellulose phthalate, methacrylicacid-methacrylic acid ester copolymers, cellulose acetate trimellitate(CAT), carboxymethyl ethyl cellulose (CMEC), hydroxypropyl methylcellulose acetate succinate (HPMCAS), and the like.

If desired, the pharmaceutical compositions of the present invention mayalso be formulated to provide slow or controlled release of the activeingredient using, by way of example, hydroxypropyl methyl cellulose invarying proportions; or other polymer matrices, liposomes and/ormicrospheres.

In addition, the pharmaceutical compositions of the present inventionmay optionally contain opacifying agents and may be formulated so thatthey release the active ingredient only, or preferentially, in a certainportion of the gastrointestinal tract, optionally, in a delayed manner.Examples of embedding compositions which can be used include polymericsubstances and waxes. The active ingredient can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically-acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Such liquid dosage formstypically comprise the active ingredient and an inert diluent, such as,for example, water or other solvents, solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (esp., cottonseed, groundnut, corn, germ,olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof. Suspensions, in addition to the active ingredient, may containsuspending agents such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Alternatively, the pharmaceutical compositions of the invention areformulated for administration by inhalation. Suitable pharmaceuticalcompositions for administration by inhalation will typically be in theform of an aerosol or a powder. Such compositions are generallyadministered using well-known delivery devices, such as a metered-doseinhaler, a dry powder inhaler, a nebulizer or a similar delivery device.

When administered by inhalation using a pressurized container, thepharmaceutical compositions of the invention will typically comprise theactive ingredient and a suitable propellant, such asdichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas.

Additionally, the pharmaceutical composition may be in the form of acapsule or cartridge (made, for example, from gelatin) comprising acompound of the invention and a powder suitable for use in a powderinhaler. Suitable powder bases include, by way of example, lactose orstarch.

The compound of the invention can also be administered transdermallyusing known transdermal delivery systems and excipients. For example,the compound of the invention can be admixed with permeation enhancers,such as propylene glycol, polyethylene glycol monolaurate,azacycloalkan-2-ones and the like, and incorporated into a patch orsimilar delivery system. Additional excipients including gelling agents,emulsifiers and buffers, may be used in such transdermal compositions ifdesired.

The following formulations illustrate representative pharmaceuticalcompositions of the present invention where compound of the inventionmeans a crystalline form of compound 1:

FORMULATION EXAMPLE A

Hard gelatin capsules for oral administration are prepared as follows:

Ingredients Amount Compound of the invention 50 mg Lactose (spray-dried)200 mg Magnesium stearate 10 mg

-   -   Representative Procedure: The ingredients are thoroughly blended        and then loaded into a hard gelatin capsule (260 mg of        composition per capsule).

FORMULATION EXAMPLE B

Hard gelatin capsules for oral administration are prepared as follows:

Ingredients Amount Compound of the invention 20 mg Starch 89 mgMicrocrystalline cellulose 89 mg Magnesium stearate 2 mg

-   -   Representative Procedure: The ingredients are thoroughly blended        and then passed through a No. 45 mesh U.S. sieve and loaded into        a hard gelatin capsule (200 mg of composition per capsule).

FORMULATION EXAMPLE C

Capsules for oral administration are prepared as follows:

Ingredients Amount Compound of the invention 10 mg Polyoxyethylenesorbitan monooleate 50 mg Starch powder 250 mg

-   -   Representative Procedure: The ingredients are thoroughly blended        and then loaded into a gelatin capsule (310 mg of composition        per capsule).

FORMULATION EXAMPLE D

Tablets for oral administration are prepared as follows:

Ingredients Amount Compound of the invention 5 mg Starch 50 mgMicrocrystalline cellulose 35 mg Polyvinylpyrrolidone (10 wt. % inwater) 4 mg Sodium carboxymethyl starch 4.5 mg Magnesium stearate 0.5 mgTalc 1 mg

-   -   Representative Procedure: The active ingredient, starch and        cellulose are passed through a No. 45 mesh U.S. sieve and mixed        thoroughly. The solution of polyvinylpyrrolidone is mixed with        the resulting powders, and this mixture is then passed through a        No. 14 mesh U.S. sieve. The granules so produced are dried at        50-60° C. and passed through a No. 18 mesh U.S. sieve. The        sodium carboxymethyl starch, magnesium stearate and talc        (previously passed through a No. 60 mesh U.S. sieve) are then        added to the granules. After mixing, the mixture is compressed        on a tablet machine to afford a tablet weighing 100 mg.

FORMULATION EXAMPLE E

Tablets for oral administration are prepared as follows:

Ingredients Amount Compound of the invention 25 mg Microcrystallinecellulose 400 mg Silicon dioxide fumed 10 mg Stearic acid 5 mg

-   -   Representative Procedure: The ingredients are thoroughly blended        and then compressed to form tablets (440 mg of composition per        tablet).

FORMULATION EXAMPLE F

Single-scored tablets for oral administration are prepared as follows:

Ingredients Amount Compound of the invention 15 mg Cornstarch 50 mgCroscarmellose sodium 25 mg Lactose 120 mg Magnesium stearate 5 mg

-   -   Representative Procedure: The ingredients are thoroughly blended        and compressed to form a single-scored tablet (215 mg of        compositions per tablet).

FORMULATION EXAMPLE G

A suspension for oral administration is prepared as follows:

Ingredients Amount Compound of the invention 0.1 g Fumaric acid 0.5 gSodium chloride 2.0 g Methyl paraben 0.15 g Propyl paraben 0.05 gGranulated sugar 25.5 g Sorbitol (70% solution) 12.85 g Veegum k(Vanderbilt Co.) 1.0 g Flavoring 0.035 mL Colorings 0.5 mg Distilledwater q.s. to 100 mL

-   -   Representative Procedure: The ingredients are mixed to form a        suspension containing 10 mg of active ingredient per 10 mL of        suspension.

FORMULATION EXAMPLE H

A dry powder for administration by inhalation is prepared as follows:

Ingredients Amount Compound of the invention 1.0 mg Lactose 25 mg

-   -   Representative Procedure: The active ingredient is micronized        and then blended with lactose. This blended mixture is then        loaded into a gelatin inhalation cartridge. The contents of the        cartridge are administered using a powder inhaler.

FORMULATION EXAMPLE I

A dry powder for administration by inhalation in a metered dose inhaleris prepared as follows:

-   -   Representative Procedure: A suspension containing 5 wt. % of a        compound of the invention and 0.1 wt. % lecithin is prepared by        dispersing 10 g of active compound as micronized particles with        mean size less than 10 μm in a solution formed from 0.2 g of        lecithin dissolved in 200 mL of demineralized water. The        suspension is spray dried and the resulting material is        micronized to particles having a mean diameter less than 1.5 μm.        The particles are loaded into cartridges with pressurized        1,1,1,2-tetrafluoroethane.

FORMULATION EXAMPLE J

An injectable formulation is prepared as follows:

Ingredients Amount Compound of the invention 0.2 g Sodium acetate buffersolution (0.4M) 40 mL HCl (0.5N) or NaOH (0.5N) q.s. to pH 4 Water(distilled, sterile) q.s. to 20 mL

-   -   Representative Procedure: The above ingredients are blended and        the pH is adjusted to 4±0.5 using 0.5 N HCl or 0.5 N NaOH.

FORMULATION EXAMPLE K

Capsules for oral administration are prepared as follows:

Ingredients Amount Compound of the Invention 4.05 mg Microcrystallinecellulose (Avicel PH 103) 259.2 mg Magnesium stearate 0.75 mg

-   -   Representative Procedure: The ingredients are thoroughly blended        and then loaded into a gelatin capsule (Size #1, White, Opaque)        (264 mg of composition per capsule).

FORMULATION EXAMPLE L

Capsules for oral administration are prepared as follows:

Ingredients Amount Compound of the Invention 8.2 mg Microcrystallinecellulose (Avicel PH 103) 139.05 mg Magnesium stearate 0.75 mg

-   -   Representative Procedure: The ingredients are thoroughly blended        and then loaded into a gelatin capsule (Size #1, White, Opaque)        (148 mg of composition per capsule).

It will be understood that even those pharmaceutical compositions inwhich the crystallinity of the compound is not maintained, for example,solution formulations, are advantageously prepared from compound 1 in acrystalline form.

Utility

The benzimidazole-carboxamide compound of the invention is a 5-HT₄receptor agonist and therefore is expected to be useful for treatingmedical conditions mediated by 5-HT₄ receptors or associated with 5-HT₄receptor activity, i.e. medical conditions which are ameliorated bytreatment with a 5-HT₄ receptor agonist. Such medical conditionsinclude, but are not limited to, irritable bowel syndrome (IBS), chronicconstipation, functional dyspepsia, delayed gastric emptying,gastroesophageal reflux disease (GERD), gastroparesis, post-operativeileus, intestinal pseudo-obstruction, and drug-induced delayed transit.In addition, it has been suggested that some 5-HT₄ receptor agonistcompounds may be used in the treatment of central nervous systemdisorders including cognitive disorders, behavioral disorders, mooddisorders, and disorders of control of autonomic function.

In particular, the compound of the invention increases motility of thegastrointestinal (GI) tract and thus is expected to be useful fortreating disorders of the GI tract caused by reduced motility inmammals, including humans. Such GI motility disorders include, by way ofillustration, chronic constipation, constipation-predominant irritablebowel syndrome (C-IBS), diabetic and idiopathic gastroparesis, andfunctional dyspepsia.

In one aspect, therefore, the invention provides a method of increasingmotility of the gastrointestinal tract in a mammal, the methodcomprising administering to the mammal a therapeutically effectiveamount of a pharmaceutical composition comprising apharmaceutically-acceptable carrier and the compound of the invention.

When used to treat disorders of reduced motility of the GI tract orother conditions mediated by 5-HT₄ receptors, the compound of theinvention will typically be administered orally in a single daily doseor in multiple doses per day, although other forms of administration maybe used. The amount of active agent administered per dose or the totalamount administered per day will typically be determined by a physician,in the light of the relevant circumstances, including the condition tobe treated, the chosen route of administration, the actual compoundadministered and its relative activity, the age, weight, and response ofthe individual patient, the severity of the patient's symptoms, and thelike.

Suitable doses for treating disorders of reduced motility of the GItract or other disorders mediated by 5-HT₄ receptors will range fromabout 0.0007 to about 20 mg/kg/day of active agent, including from about0.0007 to about 1 mg/kg/day. For an average 70 kg human, this wouldamount to from about 0.05 to about 70 mg per day of active agent.

In one aspect of the invention, the compound of the invention is used totreat chronic constipation. When used to treat chronic constipation, thecompound of the invention will typically be administered orally in asingle daily dose or in multiple doses per day. Preferably, the dose fortreating chronic constipation will range from about 0.05 to about 70 mgper day.

In another aspect of the invention, the compound of the invention isused to treat irritable bowel syndrome. When used to treatconstipation-predominant irritable bowel syndrome, the compound of theinvention will typically be administered orally in a single daily doseor in multiple doses per day. Preferably, the dose for treatingconstipation-predominant irritable bowel syndrome will range from about0.05 to about 70 mg per day.

In another aspect of the invention, the compound of the invention isused to treat diabetic gastroparesis. When used to treat diabeticgastroparesis, the compound of the invention will typically beadministered orally in a single daily dose or in multiple doses per day.Preferably, the dose for treating diabetic gastroparesis will range fromabout 0.05 to about 70 mg per day.

In yet another aspect of the invention, the compound of the invention isused to treat functional dyspepsia. When used to treat functionaldyspepsia, the compound of the invention will typically be administeredorally in a single daily dose or in multiple doses per day. Preferably,the dose for treating functional dyspepsia will range from about 0.05 toabout 70 mg per day.

The invention also provides a method of treating a mammal having adisease or condition associated with 5-HT₄ receptor activity, the methodcomprising administering to the mammal a therapeutically effectiveamount of the compound of the invention or of a pharmaceuticalcomposition comprising a compound of the invention.

As described above, the compound of the invention is a 5-HT₄ receptoragonist. The invention further provides, therefore, a method ofagonizing a 5-HT₄ receptor in a mammal, the method comprisingadministering the compound of the invention to the mammal.

Among other properties, the compound of the invention has been found tobe a potent agonist of the 5-HT₄ receptor and to exhibit exceptionalselectivity for the 5-HT₄ receptor subtype over the 5-HT₃ receptorsubtype in radioligand binding assays. Further, the compound of theinvention has demonstrated superior pharmacokinetic properties in a ratmodel. The compound is thus expected to be highly bioavailable upon oraladministration. In addition, the compound has been shown not to exhibitan unacceptable level of inhibition of the potassium ion current in anin vitro voltage-clamp model using isolated whole cells expressing thehERG cardiac potassium channel. The voltage-clamp assay is an acceptedpre-clinical method of assessing the potential for pharmaceutical agentsto change the pattern of cardiac repolarization, specifically to cause,so-called QT prolongation, which has been associated with cardiacarrhythmia. (Cavero et al., Opinion on Pharmacotherapy, 2000, 1, 947-73,Fermini et al., Nature Reviews Drug Discovery, 2003, 2, 439-447)Accordingly, pharmaceutical compositions comprising the compound of theinvention are expected to have an acceptable cardiac profile.

There properties, as well as the utility of the compound of theinvention, can be demonstrated using various in vitro and in vivo assayswell-known to those skilled in the art. Representative assays aredescribed in further detail in the following examples.

EXAMPLES

The following synthetic and biological examples are offered toillustrate the invention, and are not to be construed in any way aslimiting the scope of the invention. In the examples below, thefollowing abbreviations have the following meanings unless otherwiseindicated. Abbreviations not defined below have their generally acceptedmeanings.

Boc=tert-butoxycarbonyl

DMSO=dimethyl sulfoxide

MeCN=acetonitrile

TFA=trifluoroacetic acid

R_(f)=retention factor

Reagents and solvents were purchased from commercial suppliers (Aldrich,Fluka, Sigma, etc.), and used without further purification. Reactionswere run under nitrogen atmosphere, unless noted otherwise. Progress ofreaction mixtures was monitored by thin layer chromatography (TLC),analytical high performance liquid chromatography (anal. HPLC), and massspectrometry, the details of which are given below and separately inspecific examples of reactions. Reaction mixtures were worked up asdescribed specifically in each reaction; commonly they were purified byextraction and other purification methods such as temperature-, andsolvent-dependent crystallization, and precipitation. In addition,reaction mixtures were routinely purified by preparative HPLC: a generalprotocol is described below. Characterization of reaction products wasroutinely carried out by mass and ¹H-NMR spectrometry. For NMRmeasurement, samples were dissolved in deuterated solvent (CD₃OD, CDCl₃,or DMSO-d₆), and ¹H-NMR spectra were acquired with a Varian Gemini 2000instrument (300 MHz) under standard observation conditions. Massspectrometric identification of compounds was performed by anelectrospray ionization method (ESMS) with an Applied Biosystems (FosterCity, Calif.) model API 150 EX instrument or an Agilent (Palo Alto,Calif.) model 1100 LC/MSD instrument.

Preparation 1: Synthesis of 2-isopropyl-1H-benzoimidazole-4-carboxylicacid (piperidin-4-ylmethyl)amide a. Preparation of 2,3-diaminobenzoicacid methyl ester

To a nitrogen-saturated solution of 2-amino-3-nitrobenzoic acid methylester (Chess GmbH, 50 g, 0.26 mol) in absolute ethanol (800 mL) wasadded palladium hydroxide (Degussa, 20% w/w on carbon, 58.75% w/w water,10 g). The slurry was degassed then shaken vigorously under hydrogen (4atm) at room temperature for 48 h. The catalyst was filtered and thefiltrate concentrated in vacuo to afford 2,3-diaminobenzoic acid methylester as a dark orange oil that solidified on standing (43 g, 0.26 mol,100%). (m/z): [M-OCH₃]⁺ calcd for C₈H₁₀N₂O₂ 135.05; found 135.3. ¹H NMR(300 MHz, DMSO-d₆):

(ppm) 3.74 (s, 3H), 4.80 (br s, 1H), 6.20 (br s, 1H), 6.38 (t, 1H), 6.70(d, 1H), 7.06 (d, 1H).

b. Preparation of 2-isopropyl-1H-benzoimidazole-4-carboxylic acid

A slurry of 2,3-diaminobenzoic acid methyl ester (21.5 g, 0.13 mol) andisobutyric acid (36.2 mL, 0.39 mol) in aqueous hydrochloric acid (4M,210 mL) was stirred under reflux for 24 h to afford a homogenoussolution. The solution was cooled to 10° C. and the pH raised to 3.5using aqueous sodium hydroxide solution (4M, approx. 210 mL), whilemaintaining the temperature below 30° C. The reaction mixture wasstirred at room temperature for 2 h, cooled to 10° C., and the resultantprecipitate filtered off. The solid cake was transferred to a beaker andacetonitrile (300 mL) was added. The slurry was stirred at roomtemperature for 1 h then and filtered to afford a grey solid. The solidwas dried under vacuum to afford the title intermediate (23 g, 0.11 mol,87%). (m/z): [M+H]⁺ calcd for C₁₁H₁₂N₂O₂ calcd. 205.09; found 205.3. ¹HNMR (300 MHz, DMSO-d₆):

(ppm) 1.27 (d, 6H), 3.39 (m, 1H), 7.29 (t, 1H), 7.78 (m, 2H).

c. Preparation of4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)amino]methyl}-piperidine-1-carboxylicacid tert-butyl ester

To a solution of 2-isopropyl-1H-benzoimidazole-4-carboxylic acid (9.0 g,44.1 mmol) in anhydrous N,N-dimethylformamide (100 mL) was added4-aminomethyl-piperidine-1-carboxylic acid tert-butyl ester (9.4 g, 44.1mmol), followed by N,N-diisopropylethylamine (16.9 mL, 97.0 mmol). Thesolution was stirred for 15 min at room temperature prior to theaddition of hydroxybenzotriazole (5.9 g, 44.1 mmol),N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (8.4 g,44.1 mmol), and additional N,N-dimethylformamide (50 mL). The reactionmixture was stirred at room temperature for 16 h, diluted withdichloromethane (300 mL), and washed sequentially with 1M aqueousphosphoric acid, 1M aqueous sodium hydroxide and brine. The solution wasthen dried over sodium sulfate and concentrated in vacuo to afford brownoil which solidified upon addition of hexanes. The solid was filtered togive the title intermediate as a beige solid (13.8 g, 36.0 mmol, 78%).(m/z): [M+H]⁺ calcd for C₂₂H₃₂N₄O₃ 401.26; found 401.5; [M-Boc+H]⁺301.5. Retention time (anal. HPLC: 2-90% MeCN/H₂O over 6 min)=3.7 min.¹H NMR (300 MHz, DMSO-d₆): δ (ppm) 1.20 (m, 2H), 1.37 (s, 9H), 1.37 (s,6H), 1.72 (m, 1H), 1.75 (m, 2H), 2.73 (br s, 2H), 3.22 (septet, 1H),3.36 (m, 2H), 3.95 (m, 2H), 7.26 (t, 1H), 7.63 (d, 1H), 7.79 (d, 1H),10.11 (t, 1H).

d. Synthesis of 2-isopropyl-1H-benzoimidazole-4-carboxylic acid(piperidin-4-ylmethyl)amide

To a solution of4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)amino]methyl}-piperidine-1-carboxylicacid tert-butyl ester (10.8 g, 27.0 mmol) dissolved in dichloromethane(50 mL) at 0° C. was slowly added trifluoroacetic acid (50 mL) in 5 mLportions. The solution was allowed to warm to room temperature, stirredfor an additional 20 minutes then evaporated in vacuo. Excesstrifluoroacetic acid was removed by co-evaporation with toluene. Theresidue was then dissolved in a minimal volume of dichloromethane andslowly added to diethyl ether (1 L) at 0° C. The resulting slurry wasstirred for 2 h at room temperature then filtered to afford thebis-trifluoroacetate salt of the title compound as a light brown solid(12.7 g, 24.0 mmol, 89%). (m/z): [M+H]⁺ calcd for C₁₇H₂₄N₄O 301.21;found 301.5. Retention time (anal. HPLC: 2-50% MeCN/H₂O over 6 min)=1.65min. ¹H NMR (300 MHz, MeOD-d₃): δ (ppm) 1.59 (d, 6H), 1.60 (m, 1H), 2.03(m, 2H), 2.04 (m, 1H), 3.00 (m, 2H), 3.43 (m, 2H), 3.45 (m, 2H), 3.63(septet, 1H), 7.63 (t, 1H), 7.90 (d, 1H), 7.96 (d, 1H), 9.04 (t, 1H).

Preparation 2: Synthesis of 4-formylpiperidine-1-carboxylic acid methylester a. Preparation of 4-hydroxymethyl-piperidine-1-carboxylic acidmethyl ester

4-Hydroxymethylpiperidine (1.0 g, 8.6 mmol) was dissolved in water (15mL) and cooled to 0° C. To this solution was added dropwise a solutionof potassium carbonate (4.8 g, 34.7 mmol) in water (10 mL), followed bymethyl chloroformate (2.68 mL, 34.7 mmol). The mixture was stirredvigorously and allowed to warm to room temperature over 2 h. Afterstirring overnight (16 h), the reaction mixture was acidified with 6Maqueous hydrochloric acid and extracted with dichloromethane (3×60 mL).The extracts were combined, dried over sodium sulfate and filtered. Thefiltrate was evaporated to yield the title intermediate (1.4 g, 8.1mmol, 93%) as a colorless oil. (m/z): C₈H₁₅NO₃ calcd. 173.11; found156.2 [M−H₂O+H]⁺. ¹H NMR (300 MHz, DMSO-d₆): δ (ppm) 0.98 (m, 2H), 1.52(m, 1H), 1.63 (br d, 2H), 2.72 (br m, 2H), 3.23 (d, 2H), 3.56 (s, 3H),3.95 (br d, 2H), 4.48 (br s, 1H).

b. Synthesis of 4-formylpiperidine-1-carboxylic acid methyl ester

To a solution of oxalyl chloride (4.1 mL, 8.2 mmol) in dichloromethane(4 mL) at −78° C. was added dropwise a solution of dimethylsulfoxide(1.2 mL, 16.4 mmol) in dichloromethane (4 mL). After stirring for 5 min,a solution of 4-hydroxymethyl-piperidine-1-carboxylic acid methyl ester(1.3 g, 7.5 mmol) in dichloromethane (5 mL) was added. The resultingsolution was stirred for another 5 min, then triethylamine (5.2 mL, 37.3mmol) was added and the mixture allowed to warm to −10° C. Afterstirring for 1 h, dichloromethane (100 mL) was added, and the organiclayer was washed with 1M aqueous phosphoric acid, 1M aqueous sodiumhydroxide, and brine. The solution was dried over sodium sulfate thenevaporated to afford the title intermediate as a wheat colored oil (1.0g, 5.8 mmol, 78%). ¹H NMR (300 MHz, DMSO-d₆): δ (ppm) 1.36 (m, 2H), 1.83(m, 2H), 2.48 (br m, 1H), 2.93 (br t, 2H), 3.56 (s, 3H), 3.80 (br d,2H), 9.56 (s, 1H).

Example 1 Synthesis of4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)-amino]methyl}piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester

2-Isopropyl-1H-benzoimidazole-4-carboxylic acid(piperidin-4-ylmethyl)amide, (bis TFA salt; 1.1 g, 2.0 mmol) wassuspended in dichloromethane (20 mL) and N,N-di-isopropylethylamine(0.72 mL, 4.0 mmol) was added. When the suspension became a clearsolution, acetic acid (0.13 mL, 2.0 mmol) was added, followed by asolution of 4-formylpiperidine-1-carboxylic acid methyl ester (0.54 g,3.1 mmol) in dichloromethane (20 mL). After stirring for 5 minutes atroom temperature, sodium triacetoxyborohydride (0.628 g, 3.1 mmol) wasadded, and the reaction stirred for an additional 1 h. The aqueous layerwas then made alkaline with 1M aqueous sodium hydroxide (35 mL) andextracted with dichloromethane (2×20 mL). The combined organic layerswere washed with brine, dried over sodium sulfate and evaporated toyield crude product as a brown solid (1.41 g).

The crude product was purified via preparative HPLC (reverse phase)[gradient of 5-10-25%:5% MeCN/water (0.1% TFA) to 10% MeCN linear over10 min; 10% MeCN to 25% MeCN linear over 50 min; flow rate=15 mL/min;detection at 280 nm] to provide the title compound as the bistrifluoroacetate salt, which was then lyophilized. A mixture of 1Msodium hydroxide and dichloromethane (1:1, 100 mL) was added to thelyophilized bis trifluoroacetate salt. The organic layer was dried oversodium sulfate, filtered, and evaporated, and the resulting solid waslyophilized to provide the title compound as a white solid (0.93 g, 2mmol, 98% yield, purity 97.5%). (m/z): [M+H]⁺ calcd for C₂₅H₃₇N₅O₃456.30; found 456.3. Retention time (anal. HPLC: 2-50% MeCN/H₂O over 6min)=3.06 min. ¹H NMR (300 MHz, DMSO-d₆): 0.92 (m, 2H), 1.30 (m, 2H),1.38 (d, 6H), 1.53 (m, 1H), 1.60-1.90 (m, 7H), 2.07 (d, 2H), 2.73 (br m,2H), 2.83 (br d, 2H), 3.22 (septet, 1H), 3.33 (t, 2H), 3.56 (s, 3H),3.93 (br d, 2H), 7.23 (t, 1H), 7.62 (d, 1H), 7.77 (d, 1H), 10.10 (br s,1H).

Example 2 Synthesis of crystalline4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)-amino]methyl}piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester (Form I)

4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)-amino]methyl}piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester in amorphous solid form, prepared according to theprocess of Example 1 (300 mg) was dissolved in acetonitrile (15 mL),mixed until complete dissolution, and exposed to the atmosphereresulting in partial evaporation. Crystals were observed to havenucleated within 2 h. Chemical composition of the crystals was confirmedby ¹H NMR, liquid chromatography/mass spectrometry (LC/MS), and x-raystructure analysis. Crystalline nature of the solid product wasconfirmed by powder x-ray diffraction, differential scanningcalorimetry, and x-ray diffraction analysis of crystal structure.

Example 3 Synthesis of crystalline4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)-amino]methyl}piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester (Form I) a. Preparation of4-hydroxymethyl-piperidine-1-carboxylic acid methyl ester

4-Hydroxymethylpiperidine (47.6 g, 1.0 eq) and water (300 mL) werecharged to a flask. The resulting mixture was cooled to 0-10° C.Potassium carbonate (85.7 g, 1.5 eq) dissolved in water (150 mL) andmethyl chloro formate (38.4 mL, 1.1 eq) were added while maintaining thetemperature at below 10° C. When the addition was complete, the reactionmixture was warmed up to 20-30° C. for 1 hour. After the reaction wascomplete, dichloromethane (500 mL) was added to the reaction mixture.The organic layer was collected and washed with 1 M phosphoric acidsolution (200 mL), saturated sodium bicarbonate solution (200 mL) andsaturated sodium chloride solution (200 mL). The organic layer was driedover sodium sulfate (50 g, 1 w/w eq) and then distilled under vacuum toproduce the title intermediate. (67.0 g, 90% yield)

b. Preparation of 4-formylpiperidine-1-carboxylic acid methyl ester

4-Hydroxymethylpiperidine-1-carboxylic acid methyl ester (34.7 g, 1.0eq) was dissolved in dichloromethane and cooled to 0-10° C. A solutionof sodium bicarbonate (2.35 g, 0.14 eq) and sodium bromide (2.40 g, 0.10eq) in water (100 mL) was added over 15 min while maintaining thetemperature at 0-10° C. 2,2,6,6-Tetramethyl-1-piperidinyloxy freeradical (TEMPO) (0.32 g, 0.01 eq) was added to the mixture, followed by10-13% w/v sodium hypochlorite solution (135 mL, 1.1 eq) over 1 h withgood agitation while maintaining the temperature at 0-10° C. After thereaction was complete, the layers were separated and the organic layerwashed with water (150 mL) and dried over sodium sulfate (30 g, 1 w/weq). The solvent was removed by distillation to provide the titleintermediate. (31.0 g, 90% yield)

c. Preparation of 2-isopropyl-1H-benzoimidazole-4-carboxylic acid(piperidin-4-ylmethyl)amide

Trifluoroacetic acid (56.0 mL, 10 eq) was added to a flask containing a˜5° C. solution of4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)amino]methyl}-piperidine-1-carboxylicacid tert-butyl ester (30.0 g, 1.0 eq) in dichloromethane (300 mL) whilemaintaining the temperature below 10° C. The resulting mixture wasstirred at 20-30° C. for 2 h. When the reaction was complete,triethylamine (73.2 mL, 7.0 eq) and acetic acid (4.3 mL, 1.0 eq) wereadded to provide a solution of the title intermediate with an apparentpH of approximately 4 that was used directly in the next step.

d. Synthesis of4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)-amino]methyl}piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester

4-Formylpiperidine-1-carboxylic acid methyl ester (25.7 g, 2.0 eq) wasadded to the solution prepared in the previous step while maintainingthe temperature at 20-30° C. After stirring for 30 min, sodiumtriacetoxyborohydride (24.3 g, 1.5 eq) was added while maintaining thetemperature at 20-30° C. The reaction mixture was stirred at 20-30° C.for 30 min. After the reaction was complete, 1 M hydrochloric acid (300mL) was added to quench the reaction. The product-containing aqueouslayer was collected and washed with dichloromethane (150 mL). Theaqueous layer was treated with activated carbon (Darco G60, 6 g, 20%w/w) to remove color. The suspension was stirred for 1 hr, and thenfiltered through a bed of Celite. Dichloromethane (300 mL) was added tothe aqueous solution and the product free-based using 4 N sodiumhydroxide by adjusting the pH of the aqueous layer to 12-13. The organiclayer was collected and washed with water (300 mL). The organic layerwas distilled at 80° C. and solvent exchanged with acetonitrile (2×300mL), to remove dichloromethane and residual triethylamine. The solidswere suspended in acetonitrile (600 mL), and the mixture heated untilthe solids were dissolved (˜75° C.). The solution was cooled untilnucleation occurred (˜55-65° C.) and held for 1 h. The slurry was cooledto 20° C. over 2 h, and then to 0-5° C. over 30 min, followed bystirring at 0-5° C. for 30 min. The solids were filtered and washed withcold acetonitrile (60 mL). The wet cake was dried under vacuum at 60° C.for 6 h to provide the title compound. (28.3 g, 85% yield).

Example 4 Synthesis of crystalline4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)-amino]methyl}piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester (Form I)

4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)-amino]methyl}piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester in amorphous solid form, prepared according to theprocess of Example 1 was dispersed in the inert diluents listed in TableI below. The mixtures were exposed to the atmosphere and allowed tocompletely evaporate. The resulting solids were characterized by powderx-ray diffraction. All solids were demonstrated to be crystalline with apowder x-ray diffraction pattern consistent with that reported below inExample 7, which was obtained from the sample of Example 2.

TABLE I Crystalline Form Synthesis Compound of Formula (I) Volume ofDiluent Diluent (mg) (mL) Ether 4.60 0.230 Cyclohexane 4.87 0.486 Ethylacetate 5.67 0.284

Example 5 Synthesis of crystalline4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)-amino]methyl}piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester (Form II)

4-(4-{[(2-Isopropyl-1H-benzoimidazole-4-carbonyl)-amino]methyl}piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester in amorphous solid form (42.2 mg), was dispersed inhexane (4.22 mL) at ambient temperature to a final concentration of 10mg/mL. The solution was sonicated to disperse larger solids. After 24 hat ambient temperature (approximately 22° C.) crystallization hadoccurred. The crystalline solids were isolated via vacuum filtration,prior to analysis.

Example 6 Synthesis of crystalline4-(4-{[(2-isopropyl-1H-benzoimidazole-4-carbonyl)-amino]methyl}piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester (Form III)

4-(4-{[(2-Isopropyl-1H-benzoimidazole-4-carbonyl)-amino]methyl}piperidin-1-ylmethyl)piperidine-1-carboxylicacid methyl ester in amorphous solid form (38 mg) was dissolved in a 1:1ethanol:water solvent mixture (1.9 mL) at ambient temperature to a finalconcentration of 20 mg/mL. The solution was sonicated for 30 seconds toensure complete dissolution. The solution was then left to slowlyevaporate in an uncapped vial. After 24 h at ambient temperaturecrystallization had occurred. The crystalline solids were isolated viavacuum filtration. The filter cake was washed once with a 1:1ethanol:water solvent mixture, prior to analysis.

Example 7 Powder X-Ray Diffraction

Powder x-ray diffraction patterns were obtained with a Thermo ARL X-RayDiffractometer Model X'TRA (Thermo ARL SA, Switzerland) using Cu Kαradiation at 1.542 Å (45 kV, 40 mA) with a Si(Li) solid-state detector.The analysis was typically performed at a scan rate of 2°/min with astep size of 0.03° per point over a range of 2° to 35° in two-thetaangle. Samples, either as received or ground to a fine powder, weregently packed into a quartz insert 7.8 mm in diameter and 0.5 mm indepth designed to fit into the instrument top-loading sample cup foranalysis. The instrument calibration to within ±0.02° two-theta anglewas verified weekly by comparison with a silicon metal standard. Arepresentative PXRD pattern for the crystalline compound of Example 2(Form I), which was hand ground to a powder, is shown in FIG. 1. Arepresentative PXRD pattern for a sample of crystalline Form IIIobtained with a Rigaku diffractometer using Cu Kα (30 kV, 15 mA)radiation is shown in FIG. 5.

Example 8 X-Ray Structure Analysis

a. Form I

A chunk crystal produced in Example 2 having dimensions of0.33×0.17×0.11 mm was mounted on a glass fiber. X-ray structure data wasobtained using a Bruker SMART 6K CCD-ray area detector with windowdiameter of 13.5 cm, controlled by SMART version 5.630 software (Bruker,2003) using Cu Kα radiation. The sample to detector distance was 5.039cm. Data was collected at a temperature of −153±1° C. and was analyzedusing SHELXS version 6.14 (Bruker, 2003) software. The following latticeparameters were derived: unit cell is orthorhombic with dimensionsa=16.9053 Å, b=9.5172 Å, c=15.4659 Å; space group is Pna2₁; calculateddensity is 1.22 g/cm. Powder x-ray diffraction peaks predicted from thederived atomic positions are in excellent agreement with the observedresults obtained as described in Example 7, as shown in Table H.

TABLE II PXRD Peak Positions Observed 2θ (degrees) Predicted 2θ(degrees) 15.08 ± 0.20 15.1 ± 0.2 15.41 ± 0.20 15.6 ± 0.2 19.00 ± 0.2019.2 ± 0.2 19.70 ± 0.20 19.5 ± 0.2 23.68 ± 0.20 23.7 ± 0.2b. Form III

A chunk crystal produced by the process of Example 6 having dimensionsof 0.35×0.12×0.09 mm was analyzed by the method described above. Thefollowing lattice parameters were derived: unit cell is monoclinic withdimensions a=14.8101 Å, b=9.9985 Å, c=17.9222 Å; β=106.3020°, spacegroup is P2₁/n; calculated density is 1.23 g/cm³.

Example 9 Thermal Analysis

Differential scanning calorimetry (DSC) was performed using a TAInstruments Model Q-100 module. Data were collected and analyzed usingTA Instruments Thermal Advantage for Q Series™ software. A sample ofabout 7 mg was accurately weighed into an aluminum pan with lid. Thesample was evaluated using a linear heating ramp of 10° C./min from 5°C. to about 200° C. The DSC cell was purged with dry nitrogen duringuse.

Thermogravimetric analysis (TGA) was performed using a TA InstrumentsModel Q-500 module. Data were collected and analyzed using TAInstruments Thermal Advantage for Q Series™ software. A sample weighingabout 2 mg was placed in an aluminum pan on a platinum cradle andscanned from ambient temperature to about 300° C. with a linear heatingrate of 10° C./min. The balance and furnace chambers were purged withnitrogen during use.

Representative DSC and TGA traces for crystalline Form I (preparedaccording to the process of Example 3), Form II, and Form III materialare shown in FIGS. 2, 4, and 6, respectively.

Example 10 Dynamic Moisture Sorption Assessment

Dynamic moisture sorption (DMS) assessment was performed at 25° C. usinga VTI atmospheric microbalance, SGA-100 system (VTI Corp., Hialeah, Fla.33016). A sample size of approximately 5-10 mg was used and the humiditywas set at the ambient value at the start of the analysis. A typical DMSanalysis consisted of three scans: ambient to 2% relative humidity (RH),2% RH to 90% RH, 90% RH to 5% RH at a scan rate of 5% RH/step. The masswas measured every two minutes and the RH was changed to the next value(±5% RH) when the mass of the sample was stable to within 0.02% for 5consecutive points. A representative DMS isotherm for the crystallinecompound of Example 2 (Form I) is shown in FIG. 3.

The crystalline compound of the invention exhibits a reversiblesorption/desorption profile with a weight change of less than 0.25% overthe entire range of 2% to 90% RH and a weight change of less than 0.1%over the critical humidity range of 40% to 75% RH.

Example 11 Infrared Analysis

The infrared (IR) absorption spectrum of the crystalline compound ofExample 2 (Form I) was determined over the frequency range 4000 to 675cm⁻¹ using an Avatar 360 FT-IR spectrometer equipped with a Nicoletattenuated total reflection (ATR) sample holder. A representative IRabsorption spectrum for a sample of the crystalline compound of theinvention had significant absorption bands at 766±1, 1097±1, 1251±1,1413±1, 1449±1, 1579±1, 1609±1, 1640±1, and 1696±1 cm⁻¹.

Example 12 Solid State Stability Assessment

Samples of the crystalline compound of Form I, prepared according to theprocess of Example 3, were stored in multiple open glass vials at 40° C.and 75% RH. At specific intervals, the contents of a representative vialwas removed and analyzed by DSC, TGA, PXRD, and by HPLC for chemicalpurity. After three months of storage, there was no detectable change inthe DSC or TGA thermograms nor in the PXRD pattern. The chemical purityof the stored sample was 99.5%.

Assay 1 Radioligand Binding Assay on 5-HT_(4(c)) Human Receptors

a. Membrane Preparation 5-HT_(4(c))

HEK-293 (human embryonic kidney) cells stably-transfected with human5-HT_(4(c)) receptor cDNA (Bmax=˜6.0 pmol/mg protein, as determinedusing [³H]-GR113808 membrane radioligand binding assay) were grown inT-225 flasks in Dulbecco's Modified Eagles Medium (DMEM) containing4,500 mg/L D-glucose and pyridoxine hydrochloride (GIBCO-InvitrogenCorp., Carlsbad Calif.: Cat #11965) supplemented with 10% fetal bovineserum (FBS) (GIBCO-Invitrogen Corp.: Cat #10437), 2 mM L-glutamine and(100 units) penicillin-(100 μg) streptomycin/ml (GIBCO-Invitrogen Corp.:Cat #15140) in a 5% CO₂, humidified incubator at 37° C. Cells were grownunder continuous selection pressure by the addition of 800 μg/mLgeneticin (GIBCO-Invitrogen Corp.: Cat #10131) to the medium.

Cells were grown to roughly 60-80% confluency (<35 subculture passages).At 20-22 hours prior to harvesting, cells were washed twice and fed withserum-free DMEM. All steps of the membrane preparation were performed onice. The cell monolayer was lifted by gentle mechanical agitation andtrituration with a 25 mL pipette. Cells were collected by centrifugationat 1000 rpm (5 min).

For the membrane preparation, cell pellets were resuspended in ice-cold50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES), pH7.4 (membrane preparation buffer) (40 mL/total cell yield from 30-40T225 flasks) and homogenized using a polytron disrupter (setting 19,2×10 s) on ice. The resultant homogenates were centrifuged at 1200 g for5 min at 4° C. The pellet was discarded and the supernatant centrifugedat 40,000 g (20 min). The pellet was washed once by resuspension withmembrane preparation buffer and centrifugation at 40,000 g (20 min). Thefinal pellet was resuspended in 50 mM HEPES, pH 7.4 (assay buffer)(equivalent 1 T225 flask/1 mL). Protein concentration of the membranesuspension was determined by the method of Bradford (Bradford, 1976).Membranes were stored frozen in aliquots at −80° C.

b. Radioligand Binding Assays

Radioligand binding assays were performed in 1.1 mL 96-deep wellpolypropylene assay plates (Axygen) in a total assay volume of 400 μLcontaining 2 μg membrane protein in 50 mM HEPES pH 7.4, containing0.025% bovine serum albumin (BSA). Saturation binding studies fordetermination of K_(d) values of the radioligand were performed using[³H]-GR113808 (Amersham Inc., Bucks, UK: Cat #TRK944; specific activity˜82 Ci/mmol) at 8-12 different concentrations ranging from 0.001 nM-5.0nM. Displacement assays for determination of pK_(i) values of compoundswere performed with [³H]-GR113808 at 0.15 nM and eleven differentconcentrations of compound ranging from 10 pM-100 μM.

Test compounds were received as 10 mM stock solutions in DMSO anddiluted to 400 μM into 50 mM HEPES pH 7.4 at 25° C., containing 0.1%BSA, and serial dilutions (1:5) then made in the same buffer.Non-specific binding was determined in the presence of 1 μM unlabeledGR113808. Assays were incubated for 60 min at room temperature, and thenthe binding reactions were terminated by rapid filtration over 96-wellGF/B glass fiber filter plates (Packard BioScience Co., Meriden, Conn.)presoaked in 0.3% polyethyleneimine. Filter plates were washed threetimes with filtration buffer (ice-cold 50 mM HEPES, pH7.4) to removeunbound radioactivity. Plates were dried, 35 μL Microscint-20 liquidscintillation fluid (Packard BioScience Co., Meriden, Conn.) was addedto each well and plates were counted in a Packard Topcount liquidscintillation counter (Packard BioScience Co., Meriden, Conn.).

Binding data were analyzed by nonlinear regression analysis with theGraphPad Prism Software package (GraphPad Software, Inc., San Diego,Calif.) using the 3-parameter model for one-site competition. The BOTTOM(curve minimum) was fixed to the value for nonspecific binding, asdetermined in the presence of 1 μM GR113808. K_(i) values for testcompounds were calculated, in Prism, from the best-fit IC₅₀ values, andthe K_(d) value of the radioligand, using the Cheng-Prusoff equation(Cheng and Prusoff, Biochemical Pharmacology, 1973, 22, 3099-108):K_(i)=IC₅₀/(1+[L]/K_(d)) where [L]=concentration [³H]-GR113808. Resultsare expressed as the negative decadic logarithm of the K_(i) values,pK_(i).

Test compounds having a higher pK_(i) value in this assay have a higherbinding affinity for the 5-HT₄ receptor. The compound of the inventionhad a pK_(i) value above about 9.

Assay 2 Radioligand Binding Assay on 5-HT_(3A) Human Receptors:Determination of Receptor Subtype Selectivity

a. Membrane Preparation 5-HT_(3A)

HEK-293 (human embryonic kidney) cells stably-transfected with human5-HT_(3A) receptor cDNA were obtained from Dr. Michael Bruess(University of Bonn, GDR) (Bmax=˜9.0 pmol/mg protein, as determinedusing [³H]-GR65630 membrane radioligand binding assay). Cells were grownin T-225 flasks or cell factories in 50% Dulbecco's Modified EaglesMedium (DMEM) (GIBCO-Invitrogen Corp., Carlsbad, Calif.: Cat #11965) and50% Ham's F12 (GIBCO-Invitrogen Corp.: Cat #11765) supplemented with 10%heat inactivated fetal bovine serum (FBS) (Hyclone, Logan, Utah: Cat#SH30070.03) and (50 units) penicillin-(50 μg) streptomycin/ml(GIBCO-Invitrogen Corp.: Cat #15140) in a 5% CO₂, humidified incubatorat 37° C.

Cells were grown to roughly 70-80% confluency (<35 subculture passages).All steps of the membrane preparation were performed on ice. To harvestthe cells, the media was aspirated and cells were rinsed with Ca²⁺,Mg²⁺-free Dulbecco's phosphate buffered saline (dPBS). The cellmonolayer was lifted by gentle mechanical agitation. Cells werecollected by centrifugation at 1000 rpm (5 min). Subsequent steps of themembrane preparation followed the protocol described above for themembranes expressing 5-HT_(4(c)) receptors.

b. Radioligand Binding Assays

Radioligand binding assays were performed in 96-well polypropylene assayplates in a total assay volume of 200 μL containing 1.5-2 μg membraneprotein in 50 mM HEPES pH 7.4, containing 0.025% BSA assay buffer.Saturation binding studies for determination of K_(d) values of theradioligand were performed using [³H]-GR65630 (PerkinElmer Life SciencesInc., Boston, Mass.: Cat #NET1011, specific activity ˜85 Ci/mmol) attwelve different concentrations ranging from 0.005 nM to 20 nM.Displacement assays for determination of pK_(i) values of compounds wereperformed with [³H]-GR65630 at 0.50 nM and eleven differentconcentrations of compound ranging from 10 pM to 100 μM. Compounds werereceived as 10 mM stock solutions in DMSO (see section 3.1), diluted to400 μM into 50 mM HEPES pH 7.4 at 25° C., containing 0.1% BSA, andserial (1:5) dilutions then made in the same buffer. Non-specificbinding was determined in the presence of 10 μM unlabeled MDL72222.Assays were incubated for 60 min at room temperature, then the bindingreactions were terminated by rapid filtration over 96-well GF/B glassfiber filter plates (Packard BioScience Co., Meriden, Conn.) presoakedin 0.3% polyethyleneimine. Filter plates were washed three times withfiltration buffer (ice-cold 50 mM HEPES, pH7.4) to remove unboundradioactivity. Plates were dried, 35 μL Microscint-20 liquidscintillation fluid (Packard BioScience Co., Meriden, Conn.) was addedto each well and plates were counted in a Packard Topcount liquidscintillation counter (Packard BioScience Co., Meriden, Conn.).

Binding data were analyzed using the non-linear regression proceduredescribed above to determine K_(i) values. The BOTTOM (curve minimum)was fixed to the value for nonspecific binding, as determined in thepresence of 10 μM MDL72222. The quantity [L] in the Cheng-Prusoffequation was defined as the concentration [³H]-GR65630.

Selectivity for the 5-HT₄ receptor subtype with respect to the 5-HT₃receptor subtype was calculated as the ratioK_(i)(5-HT_(3A))/K_(i)(5-HT_(4(c))). The compound of the invention had a5-HT₄/5-HT₃ receptor subtype selectivity above about 200,000.

Assay 3 Whole-Cell cAMP Accumulation Flashplate Assay with HEK-293 CellsExpressing Human 5-HT_(4(c)) Receptors

In this assay, the functional potency of a test compound was determinedby measuring the amount of cyclic AMP produced when HEK-293 cellsexpressing 5-HT₄ receptors were contacted with different concentrationsof test compound.

a. Cell Culture

HEK-293 (human embryonic kidney) cells stably-transfected with clonedhuman 5-HT_(4(c)) receptor cDNA were prepared expressing the receptor attwo different densities: (1) at a density of about 0.5-0.6 pmol/mgprotein, as determined using a [³H]-GR113808 membrane radioligandbinding assay, and (2) at a density of about 6.0 pmol/mg protein. Thecells were grown in T-225 flasks in Dulbecco's Modified Eagles Medium(DMEM) containing 4,500 mg/L D-glucose (GIBCO-Invitrogen Corp.: Cat#11965) supplemented with 10% fetal bovine serum (FBS) (GIBCO-InvitrogenCorp.: Cat #10437) and (100 units) penicillin-(100 μg) streptomycin/ml(GIBCO-Invitrogen Corp.: Cat #15140) in a 5% CO₂, humidified incubatorat 37° C. Cells were grown under continuous selection pressure by theaddition of geneticin (800 μg/mL: GIBCO-Invitrogen Corp.: Cat #10131) tothe medium.

b. Cell Preparation

Cells were grown to roughly 60-80% confluency. Twenty to twenty-twohours prior to assay, cells were washed twice, and fed, with serum-freeDMEM containing 4,500 mg/L D-glucose (GIBCO-Invitrogen Corp.: Cat#11965). To harvest the cells, the media was aspirated and 10 mL Versene(GIBCO-Invitrogen Corp.: Cat #15040) was added to each T-225 flask.Cells were incubated for 5 min at RT and then dislodged from the flaskby mechanical agitation. The cell suspension was transferred to acentrifuge tube containing an equal volume of pre-warmed (37° C.) dPBSand centrifuged for 5 min at 1000 rpm. The supernatant was discarded andthe pellet was re-suspended in pre-warmed (37° C.) stimulation buffer(10 mL equivalent per 2-3 T-225 flasks). This time was noted and markedas time zero. The cells were counted with a Coulter counter (count above8 μm, flask yield was 1-2×10⁷ cells/flask). Cells were resuspended at aconcentration of 5×10⁵ cells/ml in pre-warmed (37° C.) stimulationbuffer (as provided in the flashplate kit) and preincubated at 37° C.for 10 min.

cAMP assays were performed in a radioimmunoassay format using theFlashplate Adenylyl Cyclase Activation Assay System with ¹²⁵I-cAMP(SMP004B, PerkinElmer Life Sciences Inc., Boston, Mass.), according tothe manufacturer's instructions.

Cells were grown and prepared as described above. Final cellconcentrations in the assay were 25×10³ cells/well and the final assayvolume was 100 μL. Test compounds were received as 10 mM stock solutionsin DMSO, diluted to 400 μM into 50 mM HEPES pH 7.4 at 25° C., containing0.1% BSA, and serial (1:5) dilutions then made in the same buffer.Cyclic AMP accumulation assays were performed with 11 differentconcentrations of compound ranging from 10 pM to 100 μM (final assayconcentrations). A 5-HT concentration-response curve (10 pM to 100 μM)was included on every plate. The cells were incubated, with shaking, at37° C. for 15 min and the reaction terminated by addition of 100 μl ofice-cold detection buffer (as provided in the flashplate kit) to eachwell. The plates were sealed and incubated at 4° C. overnight. Boundradioactivity was quantified by scintillation proximity spectroscopyusing the Topcount (Packard BioScience Co., Meriden, Conn.).

The amount of cAMP produced per mL of reaction was extrapolated from thecAMP standard curve, according to the instructions provided in themanufacturer's user manual. Data were analyzed by nonlinear regressionanalysis with the GraphPad Prism Software package using the 3-parametersigmoidal dose-response model (slope constrained to unity). Potency dataare reported as pEC₅₀ values, the negative decadic logarithm of the EC₅₀value, where EC₅₀ is the effective concentration for a 50% maximalresponse.

Test compounds exhibiting a higher pEC₅₀ value in this assay have ahigher potency for agonizing the 5-HT₄ receptor. The compound of theinvention which was tested in this assay, for example, in the cell line(1) having a density of about 0.5-0.6 pmol/mg protein, had a pEC₅₀ valueabove about 9.

Assay 4 In Vitro Voltage Clamp Assay of Inhibition of Potassium IonCurrent in Whole Cells Expressing the hERG Cardiac Potassium Channel

CHO-K 1 cells stably transfected with hERG cDNA were obtained from GailRobertson at the University of Wisconsin. Cells were held in cryogenicstorage until needed. Cells were expanded and passaged in Dulbecco'sModified Eagles Medium/F12 supplemented with 10% fetal bovine serum and200 μg/mL geneticin. Cells were seeded onto poly-D-lysine (100 μg/mL)coated glass coverslips, in 35 mm² dishes (containing 2 mL medium) at adensity that enabled isolated cells to be selected for whole cellvoltage-clamp studies. The dishes were maintained in a humidified, 5%CO₂ environment at 37° C.

Extracellular solution was prepared at least every 7 days and stored at4° C. when not in use. The extracellular solution contained (mM): NaCl(137), KCl (4), CaCl₂ (1.8), MgCl₂ (1), Glucose (10),4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES) (10), pH 7.4with NaOH. The extracellular solution, in the absence or presence oftest compound, was contained in reservoirs, from which it flowed intothe recording chamber at approximately 0.5 mL/min. The intracellularsolution was prepared, aliquoted and stored at −20° C. until the day ofuse. The intracellular solution contained (mM): KCl (130), MgCl₂ (1),ethylene glycol-bis(beta-aminoethyl ether) N,N,N′,N′-tetra acetic acidsalt (EGTA) (5), MgATP (5),4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES) (10), pH 7.2with KOH. All experiments were performed at room temperature (20-22°C.).

The coverslips on which the cells were seeded were transferred to arecording chamber and perfused continuously. Gigaohm seals were formedbetween the cell and the patch electrode. Once a stable patch wasachieved, recording commenced in the voltage clamp mode, with theinitial holding potential at −80 mV. After a stable whole-cell currentwas achieved, the cells were exposed to test compound. The standardvoltage protocol was: step from the holding potential of −80 mV to +20mV for 4.8 sec, repolarize to −50 mV for 5 sec and then return to theoriginal holding potential (−80 mV). This voltage protocol was run onceevery 15 sec (0.067 Hz). Peak current amplitudes during therepolarization phase were determined using pClamp software. Testcompounds at a concentration of 3 μM were perfused over the cells for 5minutes, followed by a 5-minute washout period in the absence ofcompound. Finally a positive control (cisapride, 20 nM) was added to theperfusate to test the function of the cell. The step from −80 mV to +20mV activates the hERG channel, resulting in an outward current. The stepback to −50 mV results in an outward tail current, as the channelrecovers from inactivation and deactivates.

Peak current amplitudes during the repolarization phase were determinedusing pCLAMP software. The control and test article data were exportedto Origin® (OriginLab Corp., Northampton Mass.) where the individualcurrent amplitudes were normalized to the initial current amplitude inthe absence of compound. The normalized current means and standarderrors for each condition were calculated and plotted versus the timecourse of the experiment.

Comparisons were made between the observed K⁺ current inhibitions afterthe five-minute exposure to either the test article or vehicle control(usually 0.3% DMSO). Statistical comparisons between experimental groupswere performed using a two-population, independent t-test (MicrocalOrigin v. 6.0). Differences were considered significant at p<0.05.

The smaller the percentage inhibition of the potassium ion current inthis assay, the smaller the potential for test compounds to change thepattern of cardiac repolarization when used as therapeutic agents. Thecompound of the invention which was tested in this assay at aconcentration of 3 μM exhibited an inhibition of the potassium ioncurrent of less than about 20%.

Assay 5 In Vitro Model of Oral Bioavailability: Caco-2 Permeation Assay

The Caco-2 permeation assay was performed to model the ability of testcompounds to pass through the intestine and get into the blood streamafter oral administration. The rate at which test compounds in solutionpermeate a cell monolayer designed to mimic the tight junction of humansmall intestinal monolayers was determined.

Caco-2 (colon, adenocarcinoma; human) cells were obtained from ATCC(American Type Culture Collection; Rockville, Md.). For the permeationstudy, cells were seeded at a density of 63,000 cells/cm² on pre-wettedtranswells polycarbonate filters (Costar; Cambridge, Mass.). A cellmonolayer was formed after 21 days in culture. Following cell culture inthe transwell plate, the membrane containing the cell monolayer wasdetached from the transwell plate and inserted into the diffusionchamber (Costar; Cambridge, Mass.). The diffusion chamber was insertedinto the heating block which was equipped with circulating external,thermostatically regulated 37° C. water for temperature control. The airmanifold delivered 95% O₂/5% CO₂ to each half of a diffusion chamber andcreated a laminar flow pattern across the cell monolayer, which waseffective in reducing the unstirred boundary layer.

The permeation study was performed with test compound concentrations at100 μM and with ¹⁴C-mannitol to monitor the integrity of the monolayer.All experiments were conducted at 37° C. for 60 min. Samples were takenat 0, 30 and 60 min from both the donor and receiver sides of thechamber. Samples were analyzed by HPLC or liquid scintillation countingfor test compound and mannitol concentrations. The permeationcoefficient (K_(p)) in cm/sec was calculated.

In this assay, a K_(p) value greater than about 10×10⁻⁶ cm/sec isconsidered indicative of favorable bioavailability. The compound of theinvention exhibited a K_(p) value greater than about 50×10⁻⁶ cm/sec.

Assay 6 Pharmacokinetic Study in the Rat

Aqueous solution formulations of test compounds were prepared in 0.1%lactic acid at a pH of between about 5 and about 6. Male Sprague-Dawleyrats (CD strain, Charles River Laboratories, Wilmington, Mass.) weredosed with test compounds via intravenous administration (IV) at a doseof 2.5 mg/kg or by oral gavage (PO) at a dose of 5 mg/kg. The dosingvolume was 1 mL/kg for IV and 2 mL/kg for PO administration. Serialblood samples were collected from animals pre-dose, and at 2 (IV only),5, 15, and 30 min, and at 1, 2, 4, 8, and 24 hours post-dose.Concentrations of test compounds in blood plasma were determined byliquid chromatography-mass spectrometry analysis (LC-MS/MS) (MDS SCIEX,API 4000, Applied Biosystems, Foster City, Calif.) with a lower limit ofquantitation of 1 ng/mL.

Standard pharmacokinetic parameters were assessed by non-compartmentalanalysis (Model 201 for 1V and Model 200 for PO) using WinNonlin(Version 4.0.1, Pharsight, Mountain View, Calif.). The maximum in thecurve of test compound concentration in blood plasma vs. time is denotedC_(max). The area under the concentration vs. time curve from the timeof dosing to the last measurable concentration (AUC(0-t)) was calculatedby the linear trapezoidal rule. Oral bioavailability (F(%)), i.e. thedose-normalized ratio of AUC(0-t) for PO administration to AUC(0-t) forIV administration, was calculated as:

F(%)=AUC_(PO)/AUC_(IV)×Dose_(IV)/Dose_(PO)×100%

Test compounds which exhibit larger values of the parameters C_(max),AUC(0-t), and F(%) in this assay are expected to have greaterbioavailability when administered orally. The compound of the inventionhad a C_(max) value of 0.8 μg/mL, an AUC(0-t) value of 1.2 μg·hr/mL andoral bioavailability (F(%)) in the rat model of about 75%.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto. Additionally, all publications, patents, andpatent documents cited hereinabove are incorporated by reference hereinin full, as though individually incorporated by reference.

1-19. (canceled)
 20. A crystalline compound of the formula

wherein the crystalline compound is characterized by a powder x-ray diffraction pattern having two or more diffraction peaks at 2θ values selected from 15.08±0.10, 15.41±0.10, 19.00±0.10, 19.70±0.10, and 23.68±0.10 and by a differential scanning calorimetry profile which shows a maximum in endothermic heat flow at a temperature in the range of about 146° C. to about 148° C.
 21. The crystalline compound of claim 20 wherein the powder x-ray diffraction pattern comprises diffraction peaks at 2θ values of 19.00±0.10 and 19.70±0.10.
 22. A crystalline compound of the formula

wherein the crystalline compound is characterized by a powder x-ray diffraction pattern in which the peak positions are substantially in accordance with the peak positions of the pattern shown in FIG. 1 and by a differential scanning calorimetry profile substantially in accordance with that shown in FIG.
 2. 23. A method of treating a disorder of reduced motility of the gastrointestinal tract in a mammal, the method comprising administering to the mammal, a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically-acceptable carrier and the crystalline compound of claim
 20. 24. The method of claim 23 wherein the disorder of reduced motility is chronic constipation, constipation-predominant irritable bowel syndrome, diabetic gastroparesis, drug-induced delayed transit, or functional dyspepsia.
 25. The method of claim 24 wherein the disorder of reduced motility is chronic constipation or constipation-predominant irritable bowel syndrome.
 26. A method of treating a disorder of reduced motility of the gastrointestinal tract in a mammal, the method comprising administering to the mammal, a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically-acceptable carrier and the crystalline compound of claim
 22. 27. The method of claim 26 wherein the disorder of reduced motility is chronic constipation, constipation-predominant irritable bowel syndrome, diabetic gastroparesis, drug-induced delayed transit, or functional dyspepsia.
 28. The method of claim 27 wherein the disorder of reduced motility is chronic constipation or constipation-predominant irritable bowel syndrome. 